ABSTRACT Traumatic brain injury (TBI) is a major public health concern affecting a large number of athletes and military personnel. Individuals suffering from a TBI risk developing anxiety disorders, yet the pathophysiological alterations that result in the development of anxiety disorders have not yet been identified. One region often damaged by a TBI is the basolateral amygdala (BLA); hyperactivity within the BLA is associated with increased expression of anxiety and fear, yet the functional alterations that lead to BLA hyperexcitability after TBI have not been identified. We assessed the functional alterations in inhibitory synaptic transmission in the BLA and one mechanism that modulates excitatory synaptic transmission, the a7 containing nicotinic acetylcholine receptor (a7-nAChR), after mTBI, to shed light on the mechanisms that contribute to increased anxiety-like behaviors. Seven and 30 days after a mild controlled cortical impact (CCI) injury, animals displayed significantly greater anxiety-like behavior. This was associated with a significant loss of GABAergic interneurons and significant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitory postsynaptic currents (IPSCs). Decreases in the mIPSC amplitude were associated with reduced surface expression of a1, b2, and c2 GABAA receptor subunits. However, significant increases in the surface expression and current mediated by a7-nAChR, were observed, signifying increases in the excitability of principal neurons within the BLA. These results suggest that mTBI causes not only a significant reduction in inhibition in the BLA, but also an increase in neuronal excitability, which may contribute to hyperexcitability and the development of anxiety disorders.

[Show abstract][Hide abstract]ABSTRACT: Neuroimaging identified abnormalities associated with traumatic brain injury (TBI) are but gross indicators that reflect underlying trauma-induced neuropathology at the cellular level. This review examines how cellular pathology relates to neuroimaging findings with the objective of more closely relating how neuroimaging findings reveal underlying neuropathology. Throughout this review an attempt will be made to relate what is directly known from post-mortem microscopic and gross anatomical studies of TBI of all severity levels to the types of lesions and abnormalities observed in contemporary neuroimaging of TBI, with an emphasis on mild traumatic brain injury (mTBI). However, it is impossible to discuss the neuropathology of mTBI without discussing what occurs with more severe injury and viewing pathological changes on some continuum from the mildest to the most severe. Historical milestones in understanding the neuropathology of mTBI are reviewed along with implications for future directions in the examination of neuroimaging and neuropathological correlates of TBI.

[Show abstract][Hide abstract]ABSTRACT: The 1991 National Health Interview Survey was analysed to describe the incidence of mild and moderate brain injury in the United States. Data were collected from 46 761 households and weighted to reflect all non-institutionalized civilians. The report of one or more occurrences of head injury resulting in loss of consciousness in the previous 12 months was the main outcome measure. Each year an estimated 1.5 million non-institutionalized US civilians sustain a non-fatal brain injury that does not result in institutionalization, a rate of 618 per 100,000 person-years. Motor vehicles were involved in 28% of the brain injuries, sports and physical activity were responsible for 20%, and assaults were responsible for 9%. Medical care was sought by 75% of those with brain injury; 14% were treated in clinics or offices, 35% were treated in emergency departments, and 25% were hospitalized. The risk of medically attended brain injury was highest among three subgroups: teens and young adults, males, and persons with low income who lived alone. The incidence of mild and moderate brain injury in the United States is substantial. The National Health Interview Survey is an important national source of current outpatient brain-injury data.

[Show abstract][Hide abstract]ABSTRACT: Nearly 64% of people with mild traumatic brain injury (MTBI) experience prolonged symptoms and functional impairments lasting months or years postinjury. Explanations for delayed recovery have varied and lacked a guiding framework, hindering intervention science. Using theory substruction and adapting McLean and associates' biopsychosocial model for chronic pain after trauma, we suggest that perceived psychological stress and associated neurobiological responses may predict risk for functional impairment. This model can be tested using a biopsychosocial approach to determine the interplay of psychological stress and neurobiological responses implicated in functional impairments after MTBI. Testing of this model will advance understanding of pathways to postconcussion syndrome.

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Reduced GABAergic Inhibition in the BasolateralAmygdala and the Development of Anxiety-LikeBehaviors after Mild Traumatic Brain InjuryCamila P. Almeida-Suhett1,4, Eric M. Prager1, Volodymyr Pidoplichko2, Taiza H. Figueiredo2,Ann M. Marini1,3,4, Zheng Li4,5, Lee E. Eiden4,6, Maria F. M. Braga1,2,4*1Program in Neuroscience, F. Edward He ´bert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America,2Department of Anatomy, Physiology and Genetics, F. Edward He ´bert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland,United States of America, 3Department of Neurology, F. Edward He ´bert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland,United States of America, 4Center for Neuroscience & Regenerative Medicine, F. Edward He ´bert School of Medicine, Uniformed Services University of the Health Sciences,Bethesda, Maryland, United States of America, 5Section on Clinical Studies, National Institute of Mental health Intramural Research Program, National Institutes of Health,Bethesda, Maryland, United States of America, 6Section on Molecular Neuroscience, National Institute of Mental health Intramural Research Program, National Institutesof Health, Bethesda, Maryland, United States of AmericaAbstractTraumatic brain injury (TBI) is a major public health concern affecting a large number of athletes and military personnel.Individuals suffering from a TBI risk developing anxiety disorders, yet the pathophysiological alterations that result in thedevelopment of anxiety disorders have not yet been identified. One region often damaged by a TBI is the basolateralamygdala (BLA); hyperactivity within the BLA is associated with increased expression of anxiety and fear, yet the functionalalterations that lead to BLA hyperexcitability after TBI have not been identified. We assessed the functional alterations ininhibitory synaptic transmission in the BLA and one mechanism that modulates excitatory synaptic transmission, the a7containing nicotinic acetylcholine receptor (a7-nAChR), after mTBI, to shed light on the mechanisms that contribute toincreased anxiety-like behaviors. Seven and 30 days after a mild controlled cortical impact (CCI) injury, animals displayedsignificantly greater anxiety-like behavior. This was associated with a significant loss of GABAergic interneurons andsignificant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitorypostsynaptic currents (IPSCs). Decreases in the mIPSC amplitude were associated with reduced surface expression of a1, b2,and c2 GABAAreceptor subunits. However, significant increases in the surface expression and current mediated by a7-nAChR, were observed, signifying increases in the excitability of principal neurons within the BLA. These results suggest thatmTBI causes not only a significant reduction in inhibition in the BLA, but also an increase in neuronal excitability, which maycontribute to hyperexcitability and the development of anxiety disorders.Citation: Almeida-Suhett CP, Prager EM, Pidoplichko V, Figueiredo TH, Marini AM, et al. (2014) Reduced GABAergic Inhibition in the Basolateral Amygdala and theDevelopment of Anxiety-Like Behaviors after Mild Traumatic Brain Injury. PLoS ONE 9(7): e102627. doi:10.1371/journal.pone.0102627Editor: Uwe Rudolph, McLean Hospital/Harvard Medical School, United States of AmericaReceived November 21, 2013; Accepted June 20, 2014; Published July 21, 2014This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone forany lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.Funding: The authors acknowledge the Department of Defense in the Center for Neuroscience and Regenerative Medicine for financially supporting the presentwork. Grant# G1702Z. URL of funder’s website: http://www.usuhs.mil/cnrm/. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* Email: maria.braga@usuhs.eduIntroductionTraumatic brain injury (TBI) is a major public health concernin the United States. There are approximately 1.8 million braininjuries annually [1], 90% of which may be classified as mild sincepatients do not display any clear morphological or functionalabnormalities following injury [2,3]. TBI is also a healthcareconcern among athletes with approximately 300,000 cases peryear [4,5]. While most patients recover fully from mild TBI(mTBI), approximately 10–15% of patients have persistentcognitive, behavioral, and emotional complaints [6–9]. Ofincreasing concern is the prevalence of anxiety disorders aftermTBI. Recent reports suggest that approximately 23% ofindividuals that sustain mTBI are at risk for developing anxietydisorders, and, particularly in the military, mTBI significantlyincreases the risk of developing posttraumatic stress disorder(PTSD) [10,11].Although clinical profiles of TBI are variable, mild andmoderate TBI have been frequently localized to medial temporallobe regions including the amygdala and are associated with long-term psychiatric symptoms [12–14]. The amygdala is a limbicstructure deep within the temporal lobe that is involved inprocessing emotion and regulating behavioral and physiologicalresponses to stressors [8,15]. Amygdalar hyperactivity has beenobserved in the majority of functional neuroimaging studiesinvestigating anxiety disorders [16]. In addition, after blastinduced TBI, bilateral amygdalar hyperactivity has been observedin U.S. soldiers [17]. Thus, amygdalar dysfunction after a TBI,and in particular, neuronal hyperexcitability and hyperactivity inthe basolateral nucleus of the amygdala (BLA), may be a keyfeature in the pathology of anxiety disorders, including PTSD inPLOS ONE | www.plosone.org1July 2014 | Volume 9 | Issue 7 | e102627

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TBI victims [10,18–21]. Although it is well established that anxietydisorders are more prevalent after mTBI, no study, to date, hasidentified the functional and morphological alterations that takeplace in the BLA underlying hyperexcitability after mTBI.Increases in anxiety-like behavior are associated with the loss ofGABAergic interneurons in the BLA [22] and reduced inhibitorysynaptic transmission [23]. However, it remains unknown whetherincreased anxiety-like behavior after mTBI is due to deficits inGABAergic inhibition in the BLA. Therefore, we examinedwhether mTBI contributes to alterations in inhibitory synaptictransmission in the BLA. To this end, we have examined whethera mild controlled cortical impact (CCI) reduces GABAAreceptormediated inhibitory postsynaptic currents (IPSCs) in the BLA anddetermined whether the loss of GABAergic inhibition is a result ofinterneuronal death or alterations in the surface expression ofGABAAreceptors. In particular, we examined the a1, b2, and c2subunits, which comprise the majority of GABAA receptorsubtypes in the brain [24]. Because a7-containing nicotinicacetylcholine receptors (a7-nAChRs) are also present in the BLAand have previously been found to modulate BLA excitability[25,26] and contribute to anxiety [27], we also examined whethera7-nAChR function and expression is altered in the BLA aftermTBI. Our results suggest that mTBI increases anxiety-likebehaviors, and that this may occur by injury-related deficits toinhibitory synaptic transmission. In addition, increased surfaceexpression and ionic flow through a7-nAChRs on glutamatergicneurons may also contribute, in part, to hyperexcitability withinthe BLA and to long lasting increases in anxiety-like behavior.Experimental ProceduresEthics StatementAll animal experiments were conducted following the Guide forthe Care and Use of Laboratory Animals (Institute of LaboratoryAnimal Resources, National Research Council) and were inaccordance with the guidelines and approved by the UniformedServices University of the Health Sciences Institutional AnimalCare and Use Committees (IACUC). All efforts were made tominimize the number of animals used and any pain or distressassociated with these experiments.AnimalsExperiments were performed using 5–6 week old male,Sprague–Dawley rats (Taconic Farms, Rockville, MD, USA).Animals were housed in pairs until the day of the surgery and thenhoused individually in an environmentally controlled room (20–23uC, ,44% humidity, 12-h light/12-h dark cycle [350–400 lux],lights on at 6:00 am), with food (Harlan Teklad Global Diet 2018,18% protein rodent diet; Harlan Laboratories; Indianapolis, IN)and water available ad libitum. Cages were cleaned weekly andanimal handling was minimized to reduce animal stress [28].Controlled Cortical Impact InjuryA unilateral cortical contusion using the controlled corticalimpact (CCI) model of traumatic brain injury was administeredusing a previously established protocol [29]. Briefly, animals wereanesthetized with isoflurane (2.5%) and had their heads shavedand placed in the stereotaxic frame. Core body temperature of theanimals was maintained at 36–37uC using a heating pad and D.C.Temperature Control System (FHC, Bowdoin, ME). Withoutdamaging the underlying dura mater, the skin was retracted, and a4.0 mm craniotomy – 3.0 mm lateral to the midline and 4.0 mmposterior to the bregma over the left tempoparietal cortex – wasperformed. In these experiments, the contact velocity was set to3.5 m/sec with a dwell time of 200 ms and the amount ofdeformation was set to 2.0 mm using a 3.0 mm diameter impacttip. These parameters have been shown to result in mild traumaticbrain injury, i.e. causing no immediate trauma-induced cell deathin areas contralateral to the impact, or even ipsilateral andsubjacent to the cortical impact area [30]. Following injury, theskullcap was replaced and fixed using bone wax (Ethicon,Sommerville, NJ) and the incision was closed with absorbablesutures (Stoelting, IL). The animals received subcutaneouslybuprenorphine (50 mL) for pain alleviation and Ringer’s solution(5 mL) for rehydration after surgery. Sham-treated controlsreceived the craniotomy, but no CCI injury.Behavioral AnalysisOpen Field Test.open field apparatus (40640620 cm clear Plexiglas arena) [31,32]2 days before and 1, 7, and 30 days after surgery. Animals wereacclimated to the apparatus in the first session. On the test day,rats were placed in the center of the open field and activity wasmeasured and recorded for 20 min, using an Accuscan Electronicsinfrared photocell system (Accuscan Instruments Inc., Columbus,OH). Data were automatically collected and transmitted to acomputer equipped with ‘‘Fusion’’ software (from AccuscanElectronics, Columbus, OH). Locomotion (distance traveled incm), total movement time, vertical activity, and time spent in thecenter of the open field were analyzed. Anxiety was measured asthe ratio of the time spent in the center over the total movementtime and expressed as a percentage of the total movement time, aspreviously described [32].Anxiety-like behavior was assessed using anImmunohistochemistryFixation and Tissue Processing.animals were deeply anesthetized using nembutal (75–100 mg/kg,i.p.) and transcardially perfused with phosphate buffered saline(PBS, 100 ml) followed by 4% paraformaldehyde (250 ml). Brainswere removed and post-fixed in 4% paraformaldehyde overnightat 4uC, then transferred to a solution of 30% sucrose in PBS for72 hours, and frozen with dry ice before storage at 280uC untilsectioning. Sectioning was performed as previously described[33,34]. A 1-in-5 series of sections containing the rostrocaudalextent of the amygdala was cut at 40 mm on a sliding microtome(Leica Microsystems SM2000R). A 1-in-5 series of free-floatingsections was collected from the cryoprotectant solution, andwashed three times for 5 min each. Slices were mounted on a slide,air-dried overnight and processed for Nissl staining with cresylviolet while the adjacent series of sections were mounted on slidesfor Fluoro-Jade C (FJC) staining.GAD-67 Immunohistochemistry.noreactive neurons, a 1-in-5 series of free-floating sections wascollected from the cryoprotectant solution, washed three times for5 min each in 0.1 M PBS, and incubated in a blocking solutioncontaining 10% normal goat serum (Millipore BioscienceResearch Reagents, Temecula, CA) and 0.5% Triton X-100 inPBS for 1 hour at room temperature. The sections were thenincubated with mouse anti-GAD-67 serum (1:1000, MAB5406;Millipore Bioscience Research Reagents), 5% normal goat serum,0.3% Triton X-100, and 1% bovine serum albumin overnight at4uC. After rinsing three times for 10 min each in 0.1% Triton X-100 in PBS, the sections were incubated with Cy3-conjugated goatanti-mouse antibody (1:1000; Jackson ImmunoResearch Labora-tories Inc., West Grove, PA) and 0.0001% 4,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich) in PBS for 1 hourat room temperature. After a final rinse in PBS for 10 min,sections were mounted on slides, air-dried for at least 30 min, andSeven (7) days after CCI,To label GAD-67 immu-Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org2July 2014 | Volume 9 | Issue 7 | e102627

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coverslipped with ProLong Gold antifade reagent (Invitrogen,Carlsbad, CA).Stereological Quantification.performed to quantify the total number of neurons in Nissl-stainedsections and interneurons in GAD-67-immunostained sections inthe BLA [34]. Sections were viewed with a Zeiss (Oberkochen,Germany) Axioplan 2ie fluorescent microscope with a motorizedstage, interfaced with a computer running StereoInvestigator 8.0(MicroBrightField, Williston, VT). The BLA was identified onslide-mounted sections and delineated for each slide of eachanimal, under a 2.56objective, based on the atlas of Paxinos andWatson [35]. All sampling was done under a 636oil immersionobjective. Nissl-stained neurons were distinguished from glia cellsby their larger size and pale nuclei surrounded by darkly stainedcytoplasm containing Nissl bodies. The total number of Nissl-stained and GAD-67-immunostained neurons was estimated byusing the optional fractionator probe, and, along with thecoefficient of error (CE), were calculated by using StereoInvesti-gator 8.0 (MicroBrightField). The CE was calculated by thesoftware according to the equations of Gundersen et al., (m=1;[36]) and Schmitz and Hof (second estimation; [37]).For Nissl-stained neurons in the BLA, a 1-in-5 series of sectionswas analyzed (six sections on average). The counting frame was35635 mm, the counting grid was 1906190 mm, and the dissectorheight was 12 mm. Nuclei were counted when the cell body cameinto focus within the dissector, which was placed 2 mm below thesection surface. Section thickness was measured at every countingsite, and the average mounted section thickness was 24.7 mm. Anaverage of 187 neurons per hemisphere per rat were counted, andthe average CE was 0.07 for both the Gunderson et al. andSchmitz-Hof equations.For GABAergic interneurons immuno-labeled for GAD-67 inthe BLA, a 1-in-5 series of sections was analyzed (on average sixsections). The counting frame was 60660 mm, the counting gridwas 1006100 mm, and the dissector height was 20 mm. Nucleiwere counted when the top of the nucleus came into focus withinthe dissector, which was placed 2 mm below the section surface.Section thickness was measured at every fifth counting site, and theaverage mounted section thickness was 24 mm. An average of 210neurons per side per rat was counted, and the average CE was0.08 for both the Gunderson et al. and Schmitz-Hof equations.Design-based stereology wasAmygdala Slice ElectrophysiologyCoronal slices containing the amygdala were prepared from rats1 or 7 days after surgery. The rats were anesthetized withisofluorane and then decapitated. Coronal brain slices (400 mm-thick) containing the amygdala (22.64 to 23.36 from Bregma)were cut using a vibratome (Leica VT 1200 S; Leica Micro-systems, Buffalo Grove, IL), in ice-cold cutting solution consistingof (in mM): 115 sucrose, 70 NMDG, 1 KCl, 2 CaCl2, 4 MgCl2,1.25 NaH2PO4, 30 NaHCO3, 25 D-glucose. Slices weretransferred to a holding chamber, maintained at 32uC for25 min, and then at room temperature, in a bath solutioncontaining (in mM): 125 NaCl, 2.5 KCl, 2.0 CaCl2, 2.0 MgCl2, 21NaHCO3, 1.25 NaH2PO4, and 22 D-glucose. Recording solutionwas the same as the holding bath solution. All solutions weresaturated with 95% O2, 5% CO2to achieve a pH near 7.4. Sliceswere transferred to a submersion-type recording chamber (0.7 mLcapacity), where they were continuously perfused with oxygenatedACSF (flow rate about 5 mL/min). Neurons were visualized withan upright microscope (Zeiss Axioskop 2, Thronwood, NY)through a 406 water immersion objective, equipped with aCCD-100 camera (Dage-MTI, Michigan City, IN). All experi-ments were performed at 32uC. Tight-seal (.1 GV) whole-cellrecordings were obtained from the cell body of pyramidal-shapedneurons in the BLA region, which were identified on the basis oftheir electrophysiological properties [38,39]. Current activated byhyperpolarization (Ihcurrent) characteristic of principal neuronswas recorded during the first minutes after breaking into the cell.Patch electrodes were fabricated from borosilicate glass and had aresistance of 3.5–4.5 MV when filled with solution A containing(in mM): 135 Cs-gluconate, 10 MgCl2, 0.1 CaCl2, 1 EGTA, 10Hepes, 2 Na–ATP, 0.2 Na2GTP, pH 7.3 (285–290 mOsm) orsolution B containing (in mM): 60 Cs-gluconate, 60 KCH3SO3, 10KCl, 10 EGTA, 10 HEPES, 5 Mg-ATP, 0.3 Na2GTP, pH 7.2(280–290 mOsm/kg). Solution A was used to record spontaneousand miniature inhibitory postsynaptic currents (IPSCs) andsolution B was used in experiments involving bath application ofcholine. Neurons were voltage-clamped using an Axopatch 200Bamplifier (Axon Instruments, Foster City, CA, USA). IPSCs werepharmacologically isolated and recorded at a 270 mV holdingpotential as inward currents in voltage-clamp mode. Accessresistance (15–24 MV) was regularly monitored during recordings,and cells were rejected if it changed by 15% during theexperiment. Ionic currents and action potentials were amplifiedand filtered (1 kHz) using the Axopatch 200B amplifier (AxonInstruments, Foster City, CA) with a four-pole, low-pass Besselfilter, were digitally sampled (up to 2 kHz) using the pClamp 10.2software (Molecular Devices, Sunnyvale, CA), and furtheranalyzed using the Mini Analysis program (Synaptosoft Inc., FortLee, NJ) and Origin (OriginLab Corporation, Northampton, MA).The peak amplitude, 10–90% rise time, and decay time constantof IPSCs were analyzed off-line using pClamp 10.2 software andthe Mini Analysis Program (Synaptosoft, Inc., Leonia, NJ, USA).Miniature IPSCs (mIPSCs) were analyzed off-line using the MiniAnalysis Program (Synaptosoft, Fort Lee, NJ) and detected bymanually setting the mIPSC threshold (,1.5 times the baselinenoise amplitude) after visual inspection.Agonists of a7-nAChRs were applied by pressure injection.Pressure application was performed with the help of a push–pullexperimental arrangement [40], as utilized previously [33].Pressure was applied to the pipette via a Picrospritzer (GeneralValve Division, Parker Hannifin Corp., Fairfield, NJ), set at about100 MPa (14 psi). A motorizer (Newport, Fountain Valley, CA)was coupled with the approach/withdrawal (push–pull) actuator ofa micromanipulator (Burleigh PCS-5000 series; EXFO PhotonicSolution Inc., Mississauga, Ontario, Canada). Motorizer move-ment and duration of application pulses were controlled with aMaster-8 digital stimulator (AMPI; Jerusalem, Israel). Ioniccurrents were amplified and filtered (1 kHz) using an Axopatch200B amplifier, with a four-pole low-pass Bessel filter, and weredigitally sampled (up to 5 kHz). Currents were recorded usingpClamp 10.2 software and further analyzed using OriginLab(Northampton, MA) and Mini60 software.Drugs used were as follows: 20 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist; 50 mM d-2-amino-phosphonovalerate (AP-5), an N-methyl-d-aspartic acid(NMDA) receptor antagonist; 10 mM SCH50911, a GABABreceptor antagonist, 3 mM LY341495, a metabotropic group II/III glutamate receptor antagonist, and 1 mM a-conotoxin Au1B,an a3b4-nicotinic receptor antagonist (all purchased from Tocris,Ellisville, MO). We also used 20 mM bicuculline methiodide, aGABAAreceptor antagonist, 1 mM tetrodotoxin (TTX), a sodiumchannel blocker, 10 mM dihydro-b-erythroidine (DHbE), an a4b2-nicotinic receptor antagonist; 5 mM tricholine citrate, an a7-nicotinic receptor agonist, and 0.5 mM atropine sulfate, aReduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org3 July 2014 | Volume 9 | Issue 7 | e102627

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Figure 1. Mild TBI increases anxiety-like behaviors in the open field test. (A) No differences in percent time spent in the center of the openfield were found between CCI (n=19) and sham (n=19) animals 24 hours after injury. However, CCI rats spent significantly less time in the center ofthe open field 7- and 30-days after injury compared to sham animals. No significant differences were found between the sham and CCI animals indistance traveled (B), vertical activity (C), or movement time (D) at any of the time points. Bars show the mean 6 SE of the percentage of time spent inthe center (A), distance traveled (B), vertical activity (C), and movement time (D). *p,0.05.doi:10.1371/journal.pone.0102627.g001Figure 2. Mild CCI does not cause a significant loss of neurons in the BLA 24 hours or 7 days after injury. (A) Panoramicphotomicrograph of Nissl-stained brain slice. Indicated are the sites of impact and the ipsilateral BLA. (B) Representative photomicrographs fromNissl-stained sections showing BLA cells from the ipsilateral (Top) and contralateral (Bottom) sides of sham, 1-day CCI, and 7-day CCI animals,respectively. Total magnification is 630X; scale bar, 50 mm. (C) Group data (mean 6 SE; n=8 for each group) of stereological estimation of the totalnumber of Nissl-stained neurons in the BLA.doi:10.1371/journal.pone.0102627.g002Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org4 July 2014 | Volume 9 | Issue 7 | e102627

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muscarinic AChR antagonist (purchased from Sigma-Aldrich, St.Louis, MO).Biotinylation and Western BlotCoronal slices containing BLA were prepared as described forelectrophysiology experiments. After a 1-hour recovery period inoxygenated ACSF, slices were incubated in ACSF containing1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) for1 hour on ice, followed by the addition of quench solution(provided in the Pierce Cell Surface Protein Isolation Kit, CatNo. 89881). The BLA was then dissected and tissue sections weretransferred to small plastic tubes containing radioimmunoprecip-itation assay (RIPA) buffer composed of (in mM) 50 Tris-HCl,pH 7.4, 150 NaCl, 2 EDTA, 50 NaF, 1 Na3VO4, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate, and a Protease InhibitorCocktail (Sigma-Aldrich, MO). Samples were sonicated and thehomogenates were centrifuged at 14,000 g for 10 min at 4uC.Protein concentrations were measured using the DC Protein AssayKit (Bio-Rad, CA). Protein (1,500 mg) was then mixed with400 mL of UltraLink immobilized NeutrAvidin agarose beads(Pierce) for 1 hour at room temperature. The beads were thenwashed 3 times with 500 mL wash buffer (provided in the kit).Samples were eluted in 400 mL of RIPA buffer containingProtease Inhibitor Cocktail supplemented with 50 mM dithiothre-itol and mixed for 1 hour at room temperature followed bycentrifugation at 14,000 g for 10 min at 4uC. Then, LDS 46(Invitrogen) was added to protein samples. Biotinylated proteinswere resolved by SDS-PAGE, transferred to nitrocellulosemembranes, and probed with the following antibodies Anti-Alpha1 GABA-A Receptor, clone N95/65, 75–136 (1:1000; UCDavis/NIH NeuroMab Facility), Anti-GABA A Receptor Beta 2,3Chain, clone BD17|MAB341 (1:1000, Millipore, Billerica, MA),Anti-GABA A Receptor Gamma 2, Ab16213 (1:1000, AbCam,Cambridge, MA), Anti-Nicotinic Acetylcholine Receptor (a7Subunit) M220 (1:1000, Sigma-Aldrich, St. Louis, MO). Thesignal from the immunoreactive band was detected by using a gelimaging system (Fuji LAS-3000). Membranes were stripped usingReBlot Plus Strong Antibody Stripping Solution (Millipore,Billerica, MA) and re-probed with Anti-GLUT1 (1:1000, Milli-pore, Billerica, MA) for loading control. Signal intensity wasdetermined by densitometric scanning using ImageJ. Whenduplicate conditions were performed in one animal, the ratiovalues were averaged to obtain an animal average for thatcondition.Statistical AnalysisStatistical values are presented as mean 6 standard error (SE).Results from ipsilateral and contralateral sides of sham-operatedand traumatized animals were compared using one-way ANOVAfollowed by Bonferroni post-hoc test in the stereology and Westernblot experiments. For open-field experiments, mixed designANOVA followed by independent t-tests were used. For electro-physiology experiments, either one-way ANOVA followed byBonferroni post-hoc test or independent t-tests were performed.p,0.05 was considered statistically significant for all statisticalanalysis. Sample sizes (n) refer to the number of rats, except for theelectrophysiology results where ‘‘n’’ refers to the number of slicesor recorded cells.ResultsAnxiety-like behaviors increase 7 days after CCIRats exposed to mild CCI were tested in the open fieldapparatus 2 days before and 1, 7 and 30 days after surgery.Overall, mTBI caused a significant increase in anxiety-likebehavior, as tested by the percent time spent in the center of theopen field (F(1,36)=4.14; p=0.049). Prior to surgery, there wereno differences between sham (8.761.2% of the total movementtime; n=19) and CCI (5.860.8% of the total movement time;n=19) groups in the percent time spent in the center of the openfield, distance traveled (2,003.46131.3 cm for sham animals,2,003.46157.0 cm for CCI animals), vertical activity (105.667.2for sham animals, 96.867.1 beam crosses for CCI animals), ormovement time (761.1620.9 s for sham animals, 742.7622.8 s forCCI animals; p’s.0.05). One day after the surgery, sham(9.361.4% of the total movement time) and CCI (10.261.7% ofthe total movement time) animals also spent similar amount oftime in the center of the field (p=0.68; Figure 1A). CCI animalsdidnotdiffer fromsham(2,034.96144.7 cm for sham animals and 2,373.76221.1 cm forCCI animals; Figure 1B), vertical activity (94.267.1 beam crossesfor sham animals, 99.169.4 beam crosses for CCI animals;Figure 1C), or movement time (754.9624.5 sec for sham animals,754.9628.2 sec; p’s.0.05; Figure 1D). However, 7 days postinjury, CCI rats (8.160.9% of the total movement time) spentsignificantly less time in the center of the open field compared tosham animals (11.461.0% of the total movement time; p=0.022).CCI animals did not differ from control animals in distancetraveled (2,605.36224.0 cm for sham animals, 2,618.06256.7 cmanimalsin distancetraveledFigure 3. Delayed loss of GABAergic interneurons in the BLA within the first week after mild CCI. (A) Representative photomicrographsof GAD-67 immunohistochemically stained GABAergic interneurons in the BLA of sham (left), 1-day CCI (middle), and 7-day CCI (right) animals. Totalmagnification is 630x; scale bar, 50 mm. (B) group data showing the mean and standard error of the stereologically estimated total number of GAD-67-positive cells in the BLA 1- and 7-days after CCI compared with sham. Only 7-days after CCI was there a significant bilateral reduction in GAD-67-positive cells indicating a delayed loss of GABAergic interneurons. ***p,0.001; n=10 for each group.doi:10.1371/journal.pone.0102627.g003Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org5 July 2014 | Volume 9 | Issue 7 | e102627

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for CCI animals), vertical activity (115.566.2 beam crossesfor sham animals, 121.967.9 beam crosses for CCI animals),or movementtime (830.9625.2 sec789.6624.6 sec for sham animals; p’s.0.05; Figure 1). Anxiety-like behavior was also observed 30 days post injury as CCI rats(9.260.8% of the total movement time) spent significantly less timein the open field compared to sham animals (14.061.9% of thetotal movement time; p=0.024; Figure 1). CCI animals did notdiffer significantly from control animals in distance traveled(2,670.36204.9 cm for sham animals, 3,165.86276.4 cm for CCIanimals), vertical activity (135.666.8 beam crosses for shamanimals, 150.968.7 beam crosses for CCI animals), or movementtime (831.2621.5 sec for sham animals, 866.6618.7 sec for shamanimals; p’s.0.05).forsham animals,Neuropathology of Principal Neurons and Interneuronsin the BLA after CCIWe next examined whether the increase in anxiety-like behaviorwas associated with a loss of neurons and interneurons after CCI.Estimation of the total number of neurons in the BLA, using anunbiased stereological method in Nissl-stained sections, revealedthat animals that received a CCI did not lose a significant numberof total neurons 1- or 7-days after injury compared to age-matchedsham injured control animals (Table 1; Figure 2). We next askedwhether CCI induced interneuronal death 1- or 7- days afterinjury, as previous studies have found a significant loss ofinterneurons after mTBI [41]. Estimation of the total number ofinterneurons in the BLA, using an unbiased stereological methodto quantify GAD67 immunoreactive cells, showed that there wasno significant loss of interneurons 1 day after CCI either ipsilateralor contralateral to the site of injury compared to sham controls(Table 2; Figure 3). Similar to the Nissl-stained sections, GAD67-positive cells from 1- and 7-day sham control groups did notdisplay any significant differences and were therefore averagedtogether (data not shown). However, 7 days after CCI, animalsshowed a 28.8% reduction ipsilateral and a 23.8% reduction inGAD67-positive cells contralateral to the site of injury, indicating asignificant loss in the number of GAD67-positive neurons in theBLA (Table 2; Figure 3B).Alterations in GABAA-mediated spontaneous andminiature IPSCsWhole-cell recordings were obtained from BLA neurons thatwere identified on the basis of their size, pyramidal-like shape,firing patterns in response to depolarizing current pulses in thecurrent-clamp mode, and the presence of a current activated byhyperpolarizing voltage-steps (Ih), in the voltage clamp mode.Depolarizing current injections generated variable patterns ofaccommodating spiking. Four 1 s-long hyperpolarizing pulsesstarting from Vhold270 to 280 mV and ending with 2110 mVelicited nonlinear Ihcurrent in principal neurons [32,42] (data notshown).To determine whether CCI impaired inhibitory synaptictransmission, we recorded spontaneous GABAAreceptor-mediat-ed IPSCs (sIPSCs) from principal neurons in the presence ofCNQX, D-AP5, SCH50911, and LY 3414953 at a holdingpotential of 270 mV. Sham animals did not differ in thefrequency and amplitude of sIPSCs at either 1- or 7-days aftersurgery and so we averaged together the amplitude and frequencyof all sham animals. We examined the frequency and amplitude ofTable 1. Total Number of Neurons.Condition# of Cells ± SEM Compared to# of Cells ± SEMSham Ipsilateral52,96961,392CCI 1 day Ipsilateral 52,81562,723CCI 1 day Contralateral 51,96161,608CCI 7 days Ipsilateral53,73362,221CCI 7 days Contralateral56,92261,451Sham Contralateral52,40061,741 CCI 1 day Ipsilateral 52,81562,723CCI 1 day Contralateral 51,96161,608CCI 7 days Ipsilateral53,73362,221CCI 7 days Contralateral56,92261,451doi:10.1371/journal.pone.0102627.t001Table 2. Total Number of Interneurons.Condition# of Cells ± SEMCompared to # of Cells ± SEMSham Ipsilateral5,5506262 CCI 1 day Ipsilateral4,76362010CCI 1 day Contralateral5,2776190CCI 7 days Ipsilateral3,9806172***CCI 7 days Contralateral4,2546107**Sham Contralateral5,7026208CCI 1 day Ipsilateral4,76362010CCI 1 day Contralateral5,2776190CCI 7 days Ipsilateral3,9806172***CCI 7 days Contralateral 4,2546107****p,0.01, ***p,0.001.doi:10.1371/journal.pone.0102627.t002Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org6July 2014 | Volume 9 | Issue 7 | e102627

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GABAA receptor-mediated sIPSCs 1- and 7-days after CCI.Compared to sham animals (mean frequency =2.960.7 Hz;mean amplitude =28869 pA; n=18), we found no significantdifference in the frequency (2.761.2 Hz; n=18; p.0.05) andamplitude (293612 pA; n=18; p.0.05) of GABAA receptor-mediated sIPSCs 1 day after CCI. However, 7 days after CCI wefound a 3269.8% reduction in the frequency (1.860.5 Hz; n=18;p,0.01) and a 2865.9% reduction in the amplitude (21168 pA;n=18; p,0.05) of GABAAreceptor mediated sIPSCs (Figure 4).To determine how CCI impaired inhibitory transmission, weexamined the effects of CCI on miniature IPSCs (mIPSCs),recorded in the presence of CNQX, D-AP5, SCH50911, and LY3414953, and TTX at a holding potential of 270 mV. RecordingmIPSCs from principal neurons in the BLA allows us to directlyexamine whether changes in the probability of quantal release atthe presynaptic terminal or internalization of postsynaptic GABAAreceptors contributed to impaired inhibitory synaptic transmission.Sham animals did not differ in the frequency and amplitude ofmIPSCs at either 1- or 7-days after surgery and so we averagedtogether the amplitude and frequency of all sham animals (datanot shown). We examined the frequency and amplitude of GABAAreceptor-mediated mIPSCs 1- and 7-days after CCI. Compared tosham animals (mean frequency =1.560.7 Hz; mean amplitude=5968 pA; n=17), we found no significant difference in thefrequency(1.560.4 Hz;n=17;(55610 pA; n=17; p.0.05) of GABAA receptor-mediatedmIPSCs 1 day after CCI. However, 7 days after CCI we founda 3565% reduction in the frequency (0.960.5 Hz; n=17; p,p.0.05)andamplitudeFigure 4. Mild CCI causes a significant decrease in the frequency and amplitude of sIPSCs in the BLA 7-days after CCI. sIPSCs wererecorded from pyramidal-shaped neurons in the presence of CNQX, D-AP5, SCH50911, and LY 3414953, at a holding potential of 270 mV.Representative examples of recordings obtained in the BLA are shown in (A) and (B) for Sham and CCI 7-day animals, respectively. (C) Group datashowing the change in the percentage frequency and amplitude of sIPSCs from CCI animals relative to Sham animals. The frequency and amplitude,but not the rise time and the decay time constant of the sIPSCs were significantly reduced in the CCI group compared to the sham controls. *p,0.05;**p,0.01; n=18 for each group.doi:10.1371/journal.pone.0102627.g004Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org7 July 2014 | Volume 9 | Issue 7 | e102627

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0.01) and a 2264.8% reduction in the amplitude (4469 pA;n=17; p,0.05) of GABAAreceptor mediated mIPSCs (Figure 5).Reduced membrane expression of GABAAreceptorsubunitsBecause we found a decrease in both the amplitude andfrequency of GABAAreceptor-mediated IPSCs, the latter of whichmay be a result of the significant loss of interneurons observed 7days after CCI, we next examined whether reduced surfaceexpression of the GABAAreceptor contributed to the reducedamplitude of mIPSCs. To determine whether the surfaceexpression of GABAAreceptor subunits was reduced after CCI,we examined alterations to the a1, b2, and c2 subunits from theBLA, subunits that constitute the majority of GABAAreceptors inthe brain and are highly expressed in the BLA [24]. Membraneproteins were isolated by biotinylation assay and levels of specificsubunits were quantified by Western blot. After densiometricanalysis, Western blot membranes were stripped and re-probed forGLUT1. It has been previously demonstrated that expression ofGLUT1 is unaltered following TBI [43] and therefore was used asa loading control. We found that the surface expression of all threeGABAA subunits were reduced 7 days (n=4) after CCI bothipsilateral (47.3%; p,0.001) and contralateral to the site of impact(52.5%; p,0.001) compared to sham control animals (n=4).Ipsilateral to the side of impact, a 48.3% (p=0.002) reduction ofthe a1 subunit, a 51.8% (p,0.001) reduction of the b2 subunit,and a 43.1% (p,0.001) reduction of the c2 subunit, were found,whereas contralateral to the side of impact, 50.2% (p,0.001)reduction of the a1 subunit, a 47.3% (p,0.001) reduction of theb2 subunit, and a 60.3% (p,0.001) reduction of the c2 subunit,was observed, indicating that the reduced amplitude in theGABAAreceptor-mediated sIPSCs may be due to reductions inthe surface expression of GABAAreceptors (Figure 6).Figure 5. Mild CCI causes a significant decrease in the frequency and amplitude of mIPSCs in the BLA 7-days after CCI. mIPSCs wererecorded from pyramidal-shaped neurons in the presence of CNQX, D-AP5, SCH50911, LY 3414953, and TTX at a holding potential of –70 mV.Representative examples of recordings obtained in the BLA are shown in (A) and (B) for Sham and CCI 7 day animals, respectively. (C) Group datashowing the change in the percentage frequency and amplitude of mIPSCs from CCI animals relative to Sham animals. The frequency and amplitude,but not the rise time and the decay time constant of the sIPSCs were significantly reduced in the CCI group compared to the sham controls. Therecorded currents were blocked by the GABAAreceptor antagonist bicuculline (data not shown). *p,0.05; **p,0.01; n=17 for each group.doi:10.1371/journal.pone.0102627.g005Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org8 July 2014 | Volume 9 | Issue 7 | e102627

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Alterations in a7-nAChR-mediated currents after mTBIIn the BLA, a7-nAChRs have previously been reported to bepresent on somatodendritic regions of glutamatergic neurons [25]and are involved in presynaptically facilitating glutamate release[26,44]. To determine whether a7-nAChRs contributed to theprincipal cell hyperexcitability, we pressure-applied tricholinecitrate (5 mM; 70 ms; 14 psi), while recording from principalneurons of sham control slices (n=17) in the presence of a-conotoxin Au1B, DHbE, atropine sulfate, D-AP5, CNQX,SCH50911, LY 3414953, and bicuculline and examined themean charge transferred, a measurement that reflects the amountof current (charged particles) flowing into the cell by integratingthe duration of the open time of ion channels. In current-clampmode, pressure application of tricholine citrate onto control sliceselicited a brief train of action potentials, while in the voltage clampmode it induced inward currents. The current induced bytricholine citrate was nearly blocked by 1 mM a-BgTx. The meancharge transferred through a7-nAChRs in sham animals was715666 pC (n=16). 1-day after CCI we did not observe anyalteration in the mean charge transferred through a7-nAChRs(748663 pC; n=16). However 7-days after CCI, the mean chargetransferred was significantly increased (970680 pC; n=17)compared to sham animals. Puff application of tricholine citrateelicited a7-nAChR currents that were 35.6% (p=0.035) larger in7-day CCI compared to sham animals, suggesting an increase inthe membrane expression of functional a7-nAChRs (Figure 7).Increased membrane expression of a7-nAChRs after CCIWe used biotinylation and Western blot analysis to determinewhether the surface expression of a7-nAChRs increase after CCIand contributed to the 35.6% increase in the charge transferred bya7-nAChRs. We found that the surface expression of the a7-nAChR was increased by 37.2% (p,0.001) 7 days after CCI(n=4) compared with sham control animals (n=8). In CCIanimals the increase in the surface expression of a7-nAChRs was44.2% (p=0.017) on the contralateral site of injury versus 37.2%(p=0.049) ipsilateral to the site of injury (Figure 8).DiscussionThe present study revealed that CCI-induced mTBI caused along-lasting increase in anxiety-like behaviors, as tested by theopen field. This was accompanied by, and may be the result ofreduced GABAAreceptor mediated inhibitory synaptic transmis-sion and increased excitability within the BLA. Reduced inhibitorytonus within the BLA is consistent with a delayed but significantloss of interneurons as compared to the total number of neuronswithin the BLA, and a significant decrease in the surfaceexpression of a1, b2, and c2 GABAAreceptors subunits aftermTBI. Interneuronal loss and reductions in the surface expressionof GABAAreceptors led to a reduction in the frequency andamplitude of GABAA-receptor mediated spontaneous and mini-ature IPSCs, respectively. Additionally, in principal neurons, weobserved a significant increase in the charge transferred by a7-nAChRs. The increase in charge transferred via the a7-nAChRswas accompanied by an increase in surface expression of a7-nAChRs in the BLA. Together, reductions in GABAergicinhibitory transmission and an increase in a7-nAChR function,may contribute, in part, to hyperexcitability in the BLA and long-lasting increases in anxiety-like behavior observed after mTBI.Long-lasting increases in anxiety and the development ofanxiety disorders have been consistently observed in humansfollowing TBI [45–47]. The recent focus of media attention onsports related injuries has revealed that mild concussions sustainedduring play increases the incidence of developing neuropsychiatricdisorders, including anxiety disorders [48,49]. Similarly, U.S.soldiers exposed to TBI are significantly more likely to reportanxiety and anxiety disorders compared to soldiers that did notsuffer a TBI [10]. Accordingly, anxiety-like behaviors have beenobserved in animals in the present study, and using differentmodels of TBI [11,50,51], including fluid percussion injury [50],blast exposure [51], and mTBI induced by CCI [11]. Thus, mTBImay cause functional and morphological alterations in the BLAunderlying long-lasting increases in anxiety-like behavior inanimals and the manifestation of anxiety disorders in humans.Although it is apparent that mild TBI increases the prevalenceof developing anxiety disorders [52], it has not been excluded thatthe anxiety may be due to the stress associated with the traumaand its aftermath and not with trauma itself [8,10,15,53]. Here, weaimed to investigate the effect of mTBI alone on the developmentof anxiety disorders. To reduce animal stress and increase thevalidity of our model, we employed strict stress-mitigatingguidelines. These included allowing animals to acclimate to theirnew environment a minimum of 3 days prior to any experimentalprocedures, cleaning cages only once per week, minimizinghandling pre- and post- surgery [28], and providing medicationto alleviate pain. Similar to other experimental procedures[11,51], animals were administered a general anesthetic prior toFigure 6. Surface expression of a1, b2, and c2 GABAAreceptor subunits is reduced in the BLA of CCI animals 7 days after mild CCI.Western blot for subunits of (A) a1, (B) b2, and (C) c2 subunits, respectively, was performed using biotinylated proteins isolated from the ipsilateraland contralateral sides of Sham and CCI 7-day animals. Group data showing the mean 6 SE of the ratio between each subunit and GLUT1 opticaldensities. Top panel: representative Western blot for a1 (A), b2 (B), and c2 (C) subunits of GABAAreceptors, respectively. Bottom panel: representativeWestern blot for GLUT1, used as a loading control. Note that surface expression of GABAAa1, b2 and c2 subunits are reduced in CCI animals whencompared to Sham animals. *p,0.01; n=4 for each group.doi:10.1371/journal.pone.0102627.g006Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org9 July 2014 | Volume 9 | Issue 7 | e102627

surgery. Our results clearly demonstrated that the increase inanxiety-like behaviors 7- and 30-days after mTBI was due to theCCI and not due to uncontrolled stress [51] or the surgery [54].Thus, while stress may be a risk factor for the development ofanxiety disorders, mTBI alone clearly caused a delayed increase inanxiety. Stress-mTBI interactions are therefore likely to mutuallyexacerbate the development of long-term symptoms of anxiety.Anxiety disorders in humans have often been attributed todeficits to the GABAergic system [55-58] but also neuronalhyperexcitability in the amygdala [59,60]. In agreement, wedemonstrate that after mTBI, there is a significant decrease ininhibitory synaptic transmission in the BLA, including reductionsin both the frequency and amplitude of mIPSCs. This reduction isconsistent with the loss of GABAergic interneurons and decreasedsurface expression of GABAA receptors, both of which areassociated with increased anxiety-like behaviors [22,61]. Ourevidence that increases in anxiety-like behavior are associated withdeficits to the GABAergic system in the BLA is corroborated byprevious studies demonstrating that ablation of a portion ofGABAergic interneurons in the BLA [22] and reduced expressionof a1 and a2 GABAAreceptor subunits within the amygdala[62,63] contributes to anxiogenic behaviors in animals. Moreover,the evidence that mTBI causes reductions in the surface expressionof a1, b2, and c2 subunits of the GABAAreceptor suggests thatthere might be decreases in the benzodiazepine binding site [24].Indeed, our evidence finding reductions in GABAergic synaptictransmission and reduced surface expression of specific subunits ofthe GABAAreceptor, provide the first functional evidence that apossible reason why many patients suffering from anxiety disordersdo not improve when treated with benzodiazepines [64–66] isbecause the GABAergic system is damaged, rendering thesecompounds less effective in mitigating anxiety-like behaviors.Excitability within the BLA is also regulated, in part, byactivation of a7-nAChRs [25,67]. a7-nAChRs are highly expressedin the BLA [25,44], play an important role in modulating bothGABAergic [68] and glutamatergic synaptic transmission, [26],and induce both anxiolytic [69] and anxiogenic effects whenactivated by specific agonists [27]. While the primary role of a7-nAChR activation appears to be an enhancement of inhibitorysynaptic transmission [68], a7-nAChR activation also enhancesexcitatory synaptic transmission [25,26,44,68]. Here, we exam-ined, for the first time, how mTBI alters a7-nAChR mediatedenhancement of principal cell excitability within the BLA. Within7 days of TBI, we found a significant increase in the surfaceexpression of a7-nAChRs and associated increases in the currentmediated by a7-nAChRs on principal neurons. Thus, we showthat in addition to a significant loss of interneurons and reductionin the inhibitory synaptic transmission, an enhancement in the a7-nAChRs-mediated transmission contributed, in part, to neuronalhyperexcitability and long-lasting increases in anxiety-like behav-ior.Is mTBI more than just a focal injury? We induced injury byplacing a single mild focal impact to the left parietal cortex, yetpathological and pathophysiological alterations in the BLA, abrain region far from the impact site, was observed on eachhemisphere. Thus, our study corroborates with the notion thatcontrolled cortical impact is actually more than a focal brain injury[70,71]. Following the impact, intracranial pressure changes oneach side of the brain, though the contralateral side is less affectedthan the ipsilateral side [72]. We speculate that such mechanicalalterations trigger a neuropathological cascade throughout thebrain that ultimately leads to the pathological and pathophysio-logical alteration observed. We recently reported that within 24hours of CCI, chemokines transcripts are upregulated and remainselevated for at least one week [30]. Subsequent inflammation takesplace and may be the underlying cause for functional andmorphological alterations in the BLA on both hemispheres herereported.In conclusion, the results from this study demonstrated that amild TBI leads to long lasting increases in anxiety-like behavior.This increase is a result of a significant loss of GABAergicinterneurons, reduced surface expression of GABAAreceptors andinhibitory synaptic transmission, and, increases in the surfaceexpression and function of a7-nAChRs, which subsequentlyincrease excitability within principal neurons in the BLA. Withthe high incidence of anxiety disorders being reported after TBI[47], it is essential to understand the pathophysiological mecha-nisms underlying their development. Although the resultsFigure 7. Activation of a7-nAChRs by fast application of the a7-nAChR agonist tricholine citrate, in the BLA, shows increasedcholinergic conductance 7-days after CCI. (A) Group data showing the mean 6 SE charge transfer in pyramidal-shaped neurons in the BLA fromCCI rats 7-days after injury (970680 pC; n=17) was significantly increased compared to sham rats (715666 pC; n=16). (B) Representative chargetransfer from a7-nAChRs from sham (left), and 7-day CCI (right) animals. Note the increase in decay through a7-nAChRs in the BLA at day 7 post injury.(C) Representative a7-nAChR-mediated currents recorded from sham (left), and 7-day CCI (right) animals. The increase in the decay and amplitude ofthe a7-nAChR-mediated current 7-days post CCI led to increases in the charge transferred through by a7-nAChRs. Experiments were recorded in thepresence of a-conotoxin Au1B, DHbE, atropine sulfate, D-AP5, CNQX, SCH50911, LY 3414953, and bicuculline. *p,0.05;doi:10.1371/journal.pone.0102627.g007Figure 8. Surface expression of a7 subunit of neuronalnicotinic acetylcholine receptor is increased in the BLA of CCIanimals 7-days after mild CCI. Western blot was performed usingbiotinylated proteins isolated from the ipsilateral and contralateral sidesof Sham and CCI 7-day animals. Group data showing the mean 6 SE ofthe ratio between the a7 subunit and GLUT1 optical densities. Toppanel: representative Western blot for a7-nAChRs. Bottom panel:representative Western blot for GLUT1, used as a loading control.*p,0.05; n=4 for each group.doi:10.1371/journal.pone.0102627.g008Reduced Inhibition in the BLA after Mild TBIPLOS ONE | www.plosone.org11 July 2014 | Volume 9 | Issue 7 | e102627